U.S. patent application number 16/614141 was filed with the patent office on 2020-05-14 for theranostic agents.
The applicant listed for this patent is THE HONG KONG UNIVERSITY OF SCIENCE AND TECHNOLOGY. Invention is credited to Dan DING, Ji QI, Benzhong TANG.
Application Number | 20200147078 16/614141 |
Document ID | / |
Family ID | 64273355 |
Filed Date | 2020-05-14 |
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United States Patent
Application |
20200147078 |
Kind Code |
A1 |
TANG; Benzhong ; et
al. |
May 14, 2020 |
THERANOSTIC AGENTS
Abstract
A theranostic agent can be used in both photoacoustic imaging
(PAI) and photothermal therapy (PTT) applications. The theranostic
agent can include a small molecule, organic compound with
absorption in the near-infrared (NIR) interrogation window (700-900
nm). The compound can be a biocompatible organic nanoparticle
(ONP). The theranostic agent can be effectively used in PAI and
PAI-guided PTT applications. The theranostic agent can be
administered to a patient to locate a tumor site in the patient
using in vivo imaging techniques. Once the tumor site has been
determined, the tumor site can be irradiated with near-infrared
light to stop or inhibit the growth of the tumor.
Inventors: |
TANG; Benzhong; (Hong Kong,
CN) ; DING; Dan; (Hong Kong, CN) ; QI; Ji;
(Hong Kong, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE HONG KONG UNIVERSITY OF SCIENCE AND TECHNOLOGY |
Hong Kong |
|
CN |
|
|
Family ID: |
64273355 |
Appl. No.: |
16/614141 |
Filed: |
May 14, 2018 |
PCT Filed: |
May 14, 2018 |
PCT NO: |
PCT/CN2018/086716 |
371 Date: |
November 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62603057 |
May 17, 2017 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 213/20 20130101;
C07D 513/04 20130101; C09K 11/06 20130101; C07D 213/24 20130101;
A61K 31/4985 20130101; A61P 35/00 20180101; A61K 49/22 20130101;
A61K 41/0052 20130101; A61K 49/221 20130101 |
International
Class: |
A61K 31/4985 20060101
A61K031/4985; A61K 41/00 20200101 A61K041/00; A61K 49/22 20060101
A61K049/22 |
Claims
1. A theranostic agent, comprising a compound having: a donor unit
selected from the group consisting of: ##STR00024## ##STR00025##
##STR00026## and an acceptor unit (A) selected from the group
consisting of: ##STR00027## wherein D and D' represent the donor
unit; wherein the compound is arranged in a form selected from the
group consisting of D-A, D-A-D, A-D-A, D-D-A-D-D, A-A-D-A-A,
D-A-D-A-D, A-D-A-D-A, wherein A represents the acceptor unit;
wherein each of X and X' is selected from the group consisting of
O, S, Se, and Te; wherein each of R, R', R'' R''', or R'''' is
unsubstituted or substituted and is selected from the group
consisting of F, H, alkyl, unsaturated alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group,
amino group, sulfonic group, alkylthio, and alkoxy group; and
wherein at least one of R, R', R'' R''', or R'''' is a terminal
functional group having a substituent independently selected from
the group consisting of N.sub.3, NCS, SH, NH.sub.2, COOH, alkyne,
N-hydroxysuccinimide ester, maleimide, hydrazide, nitrone group,
--CHO, --OH, halide, and a charged ionic group; and wherein at
least one of R, R', R'' R''', and R'''' is other than H.
2. The theranostic agent according to claim 1, wherein the compound
further comprises one or more peptides conjugated thereto.
3. The theranostic agent according to claim 1, wherein the compound
comprises a structural formula selected from the group consisting
of: ##STR00028## wherein each of X and X' is selected from the
group consisting of O, S, Se, and Te; wherein each of D and D' is
selected from the group consisting of ##STR00029## ##STR00030##
wherein each of R, R', R'', and R''' is unsubstituted or
substituted, and is selected from the group consisting of F, H,
alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group,
sulfonic group, alkylthio, and alkoxy group; wherein at least one
of R, R', R'', and R''' is a terminal functional group having a
substituent independently selected from the group consisting of
N.sub.3, NCS, SH, NH.sub.2, COOH, alkyne, N-hydroxysuccinimide
ester, maleimide, hydrazide, nitrone group, --CHO, --OH, halide,
and a charged ionic group; and wherein at least one of R, R', R'',
and R''' is other than H.
4. The theranostic agent according to claim 3, wherein the compound
further comprises one or more peptides conjugated thereto.
5. The theranostic agent according to claim 3, wherein the compound
comprises the following structural formula: ##STR00031## wherein X
is selected from the group consisting of O, S, Se, and Te; wherein
each of R, R' R'', R''', R'''' R''''', R'''''', and R''''''' is
unsubstituted or substituted, and is selected from the group
consisting of F, H, alkyl, unsaturated alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group,
amino group, sulfonic group, alkylthio, and alkoxy group; wherein
at least one of R, R', R'' R''', R'''', R''''', R'''''', and
R''''''' is a terminal functional group having a substituent
independently selected from the group consisting of N.sub.3, NCS,
SH, NH.sub.2, COOH, alkyne, N-Hydroxysuccinimide ester, maleimide,
hydrazide, nitrone group, --CHO, --OH, halide, and a charged ionic
group; and wherein at least one of R, R' R'', R''', R'''' R''''',
R'''''', and R''''''' is other than H.
6. The theranostic agent according to claim 5, wherein the compound
further comprises one or more peptides conjugated thereto.
7. The theranostic agent according to claim 3, wherein the compound
comprises the following structural formula: ##STR00032## wherein
each of R, R' R'', R''', R'''', R''''', R'''''', and R''''''' is
unsubstituted or substituted, and is selected from the group
consisting of F, H, alkyl, unsaturated alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group,
amino group, sulfonic group, alkylthio, and alkoxy group; wherein
at least one of R, R', R'', R''', R'''', R''''', R'''''', and
R''''''' is a terminal functional group having a substituent
independently selected from the group consisting of N.sub.3, NCS,
SH, NH.sub.2, COOH, alkyne, N-Hydroxysuccinimide ester, maleimide,
hydrazide, nitrone group, --CHO, --OH, halide, and a charged ionic
group; and wherein at least one of R, R', R'' R''', R'''', R''''',
R'''''', and R''''''' is other than H.
8. The theranostic agent according to claim 7, wherein the compound
further comprises one or more peptides conjugated thereto.
9. The theranostic agent according to claim 3, wherein the compound
is: ##STR00033##
10. A method of locating a tumor site in a patient, comprising:
administering the compound of claim 1 to the patient; and locating
the tumor site using photoacoustic imaging.
11. The method of claim 10, wherein the compound is administered by
intravenous injection.
12. The method of claim 10, wherein the compound is administered in
nanoparticle form.
13. A method of stopping or inhibiting tumor growth in a patient,
comprising: administering the compound of claim 1 to the patient;
locating a tumor site using photoacousting imaging; and subjecting
the tumor site to near-infrared light irradiation while the
compound is present at the tumor site to stop or inhibit the growth
of the tumor.
14. A theranostic agent, comprising a compound having the following
structural formula: ##STR00034## wherein each of R, R' R'', R''',
R'''', R''''', R'''''', and R''''''' is unsubstituted or
substituted, and is selected from the group consisting of F, H,
alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group,
sulfonic group, alkylthio, and alkoxy group; wherein at least one
of R, R', R'', R''', R'''', R''''', R'''''', and R''''''' is a
terminal functional group having a substituent independently
selected from the group consisting of N.sub.3, NCS, SH, NH.sub.2,
COOH, alkyne, N-Hydroxysuccinimide ester, maleimide, hydrazide,
nitrone group, --CHO, --OH, halide, and a charged ionic group; and
wherein at least one of R, R', R'' R''', R'''', R''''', R'''''',
and R''''''' is other than H.
15. The theranostic agent according to claim 14, wherein the
compound is: ##STR00035##
16. A method of locating a tumor site in a patient, comprising:
administering the compound of claim 14 to the patient; and locating
the tumor site using photoacoustic imaging.
17. The method of claim 17, wherein the compound is administered by
intravenous injection.
18. The method of claim 17, wherein the compound is administered in
nanoparticle form.
19. A method of stopping or inhibiting tumor growth in a patient,
comprising: administering the compound of claim 14 to the patient;
locating a tumor site using photoacousting imaging; and subjecting
the tumor site to near-infrared light irradiation while the
compound is present at the tumor site to stop or inhibit the growth
of the tumor.
Description
FIELD
[0001] The present subject matter relates generally to a series of
organic, small molecule compounds with absorption in the
near-infrared (NIR) interrogation window (700-900 nm) and their
applications in photoacoustic imaging (PAI) and photothermal
therapy (PTT).
BACKGROUND
[0002] The emergence of photo-theranostic agents has opened a new
door for cancer research. Theranostic agents can facilitate
integration of real-time diagnosis and in-situ phototherapeutic
capabilities in one platform. Among versatile light-triggered
diagnostic/therapeutic techniques, photoacoustic imaging (PAI)
associated with photothermal therapy (PTT) is particularly
effective in accurately probing tumor location and effectively
inhibiting tumor growth, with minimal side effect to normal tissue.
PAI is a very promising noninvasive molecular imaging approach that
combines deep tissue penetration and high resolution of ultrasound
imaging with high contrast of optical imaging. The therapeutic
technique that typically accompanies PAI is PTT, as PAI is used
primarily to detect the photothermally generated ultrasound signal.
The most vital prerequisite of PAI/PTT applications is to employ
efficient contrast agents with strong absorption in the
near-infrared (NIR) interrogation window (700-900 nm), since NIR
light is known to penetrate much deeper tissue and cause less
photodamage to a living body.
[0003] A variety of nanomaterials, such as metal nanomaterials
(e.g., gold, and silver nanostructures), carbon nanomaterials
(e.g., carbon nanotubes, and graphene), transition metal
dichalcogenides (e.g., MoS.sub.2, WS.sub.2, and Ag.sub.2S), and
organic material-based nanoparticles, have been extensively
investigated as PAI/PTT agents. Unlike inorganic nanoagents,
organic materials, e.g., polymers and small molecules, offer
advantages of outstanding biocompatibility, potential
biodegradability, and easy processability. Accordingly,
semiconducting polymer nanoparticles (SPNs) have recently been
explored as contrast agents for PAI, as well as PTT applications
with superb performance. However, development of organic small
molecules applicable for PAI/PTT has been less extensive even
though organic small molecules have the advantage of a well-defined
chemical structure, high purity, good reproducibility, facile
modification, and easy processibility. One challenge typically
associated with some organic small molecules involves instability
of the molecules in PAI/PTT applications, which to date has limited
development in this area.
[0004] Some conventional cyanine dyes have been investigated and
used as intermediates for light-mediated biomedical applications in
clinics. For example, indocyanine green (ICG), an ionic compound
with strong absorption in the NIR spectral region of 700-850 nm,
has been approved by the Food and Drug Administration (FDA) for
clinical use, highlighting the potential of organic small molecules
for clinical translation and practical applications. These cyanine
dyes, however, suffer from the problems of modification difficulty
and poor stability, which may lead to safety problems and untrusted
theranostic outcomes. For example, many cyanine dyes are prone to
decomposition by reactive oxygen/nitrogen species (RONS). As such,
many cyanine dyes are useful as sensitive probes for detecting RONS
in a living body. The alternatively arranged single and double
bonds in cyanine dyes are easily oxidized by the highly reactive
RONS, which results in the decrease or disappearance of featured
NIR absorption and fluorescence signals. While utilization of the
reactive feature of cyanine dyes for ratiometric sensing of RONS is
reasonable, the instability of cyanine dyes would create serious
problems in PAI/PTT applications, such as misleading PAI signals,
impaired PTT treatment efficacy, and harmful side effects caused by
in vivo decomposition.
[0005] Many of the currently available NIR-absorbing organic small
molecules face various challenges, including, photothermal
instability, photobleaching, and susceptibility to RONS
decomposition.
[0006] Accordingly, the development of highly stable NIR organic
small molecular agents for effective PAI/PTT applications is
desired.
SUMMARY
[0007] The present subject matter relates to a theranostic agent
that can be used in both photoacoustic imaging (PAI) and
photothermal therapy (PTT) applications. The theranostic agent can
include a small molecule, organic compound with absorption in the
near-infrared (NIR) interrogation window (700-900 nm). The compound
can be a biocompatible organic nanoparticle (ONP). The theranostic
agent can be administered to a patient to locate a tumor site in
the patient using photoacoustic imaging. Once the tumor site has
been determined, the tumor site can be irradiated with
near-infrared light which, when combined with the present
compounds, can stop or inhibit the growth of the tumor.
[0008] In an embodiment, the compound has:
a donor unit selected from the group consisting of
##STR00001## ##STR00002## ##STR00003##
and
[0009] an acceptor unit (A) selected from the group consisting
of:
##STR00004##
[0010] wherein D and D' represent the donor unit;
[0011] wherein the compound has a structural arrangement in a form
selected from the group consisting of D-A, D-A-D, A-D-A, D-D-A-D-D,
A-A-D-A-A, D-A-D-A-D, A-D-A-D-A,
[0012] wherein A represents the acceptor unit;
[0013] wherein each of X and X' is selected from the group
consisting of O, S, Se, and Te;
[0014] wherein each of R, R', R'' R''', or R'''' is unsubstituted
or substituted and is selected from the group consisting of F, H,
alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group,
sulfonic group, alkylthio, and alkoxy group; and
[0015] wherein at least one of R, R', R'' R''', or R'''' is a
terminal functional group having a substituent independently
selected from the group consisting of N.sub.3, NCS, SH, NH.sub.2,
COOH, alkyne, N-hydroxysuccinimide ester, maleimide, hydrazide,
nitrone group, --CHO, --OH, halide, and a charged ionic group;
and
[0016] wherein at least one of R, R', R'' R''', and R'''' is other
than H.
[0017] In a further embodiment, the compound has one of the
following structural formulae:
##STR00005##
[0018] wherein each of X and X' is selected from the group
consisting of O, S, Se, and Te;
[0019] wherein each of D and D' is selected from the group
consisting of
##STR00006## ##STR00007##
[0020] wherein each of R, R', R'', and R''' is unsubstituted or
substituted, and is selected from the group consisting of F, H,
alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group,
sulfonic group, alkylthio, and alkoxy group;
[0021] wherein at least one of R, R', R'', and R''' is a terminal
functional group having a substituent independently selected from
the group consisting of N.sub.3, NCS, SH, NH.sub.2, COOH, alkyne,
N-hydroxysuccinimide ester, maleimide, hydrazide, nitrone group,
--CHO, --OH, halide, and a charged ionic group; and
[0022] wherein at least one of R, R', R'', and R''' is other than
H.
[0023] In an embodiment, the compound has the following structural
formula:
##STR00008##
[0024] wherein X is selected from the group consisting of O, S, Se,
and Te;
[0025] wherein each of R, R' R'', R''', R'''' R''''', R'''''', and
R''''''' is unsubstituted or substituted, and is selected from the
group consisting of F, H, alkyl, unsaturated alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group,
amino group, sulfonic group, alkylthio, and alkoxy group;
[0026] wherein at least one of R, R', R'' R''', R'''', R''''',
R'''''', and R''''''' is a terminal functional group having a
substituent independently selected from the group consisting of
N.sub.3, NCS, SH, NH.sub.2, COOH, alkyne, N-Hydroxysuccinimide
ester, maleimide, hydrazide, nitrone group, --CHO, --OH, halide,
and a charged ionic group; and
[0027] wherein at least one of R, R' R'', R''', R'''' R''''',
R'''''', and R''''''' is other than H.
[0028] In an embodiment, the compound has the following structural
formula:
##STR00009##
[0029] wherein each of R, R' R'', R''', R'''', R''''', R'''''', and
R''''''' is unsubstituted or substituted, and is selected from the
group consisting of F, H, alkyl, unsaturated alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group,
amino group, sulfonic group, alkylthio, and alkoxy group;
[0030] wherein at least one of R, R', R'', R''', R'''', R''''',
R'''''', and R''''''' is a terminal functional group having a
substituent independently selected from the group consisting of
N.sub.3, NCS, SH, NH.sub.2, COOH, alkyne, N-Hydroxysuccinimide
ester, maleimide, hydrazide, nitrone group, --CHO, --OH, halide,
and a charged ionic group; and
[0031] wherein at least one of R, R', R'' R''', R'''', R''''',
R'''''', and R''''''' is other than H.
[0032] In an embodiment, an exemplary compound is:
##STR00010##
BRIEF DESCRIPTION OF DRAWINGS
[0033] Various embodiments will now be described in detail with
reference to the accompanying drawings.
[0034] FIG. 1 depicts the .sup.1H NMR spectrum of compound 10 in
CDCl.sub.3.
[0035] FIG. 2 depicts the .sup.13C NMR spectrum of compound 10 in
CDCl.sub.3.
[0036] FIG. 3 depicts the HRMS of compound 10.
[0037] FIG. 4 depicts the .sup.1H NMR spectrum of compound TPA-T-TQ
in CDCl.sub.3.
[0038] FIG. 5 depicts the .sup.13C NMR spectrum of compound
TPA-T-TQ in CDCl.sub.3.
[0039] FIG. 6 depicts the HRMS of compound TPA-T-TQ.
[0040] FIG. 7 depicts the Thermogravimetric analysis (TGA) curve of
TPA-T-TQ.
[0041] FIG. 8A depicts a schematic for preparing the organic small
molecular nanoparticles through nanoprecipitation method.
[0042] FIG. 8B depicts the UV-vis-NIR absorption spectra of
TPA-T-TQ in THF solution and the encapsulated ONPs in water.
[0043] FIG. 8C depicts the photoluminescence (PL) spectra of
TPA-T-TQ in THF solution and the encapsulated ONPs in water.
[0044] FIG. 8D depicts the TEM image of the TPA-T-TQ ONPs.
[0045] FIG. 8E depicts the DLS profile of the TPA-T-TQ ONPs.
[0046] FIG. 9A depicts IR thermal images of the ONPs and ICG in PBS
solutions (100 .mu.M) under 808-nm laser irradiation (0.8
W/cm.sup.2) for different times.
[0047] FIG. 9B depicts a graph showing photothermal conversion
behavior of TPA-T-TQ ONPs at different concentrations (5-100 .mu.M)
under 808 nm light irradiation at a power intensity of 0.8
W/cm.sup.2.
[0048] FIG. 9C depicts a graph comparing the photothermal
conversion behavior of TPA-T-TQ ONPs and ICG in PBS solution at the
same concentration (100 .mu.M) (the 808-nm light (0.8 W/cm.sup.2)
was irradiated for 5 min).
[0049] FIG. 9D depicts photographs of the ONPs and ICG in PBS
solutions after 808 nm light irradiation for different times.
[0050] FIG. 9E depicts a plot of I/I.sub.0 versus various
irradiation time (I and I.sub.0 are the maximal NIR absorption
intensity of ONPs/ICG in PBS solutions after and before laser
irradiation, respectively).
[0051] FIG. 9F depicts a plot showing anti-photobleaching property
of ONPs and ICG (100 .mu.M) during five circles of heating-cooling
processes (the laser used for irradiation was 808-nm light with a
power density of 0.8 W/cm.sup.2).
[0052] FIG. 9G depicts absorption spectra of TPA-T-TQ ONPs in PBS
solution before and after 400 .mu.M of ONOO.sup.- and *OH were
added for 1 min (inset shows photograph of the solution of ONPs
before and after the addition of RONS).
[0053] FIG. 9H depicts absorption spectra of ICG in PBS solution
before and after 400 .mu.M of ONOO.sup.- and *OH were added for 1
min (inset shows photograph of the solution of ICG before and after
the addition of RONS).
[0054] FIG. 9I depicts a plot of I/I.sub.0 versus RONS (ONOO.sup.-
and *OH). I and I.sub.0 are the maximal NIR absorption intensity of
ONPs/ICG in PBS solutions in the presence and absence of RONS,
respectively.
[0055] FIG. 10 depicts the photoluminescence (PL) spectra of ICG in
water before and after the addition 0.4 mM of ONOO.sup.- and *OH
for 1 min.
[0056] FIG. 11A depicts the PA spectrum of TPA-T-TQ ONPs in PBS
solution (110 .mu.g/mL based on TPA-T-TQ).
[0057] FIG. 11B depicts PA amplitudes of TPA-T-TQ ONPs at 770 nm as
a function of TPA-T-TQ concentration.
[0058] FIG. 11C depicts PA intensity at the tumor site as a
function of post-injection time.
[0059] FIG. 11D depicts PA images of tumor site after systemic
administration of TPA-T-TQ ONPs for designated time intervals.
[0060] FIG. 12A depicts IR thermal images of 4T1 tumor-bearing mice
under 808 nm laser irradiation (0.5 W/cm2) for different time
points.
[0061] FIG. 12B depicts a graph showing the mean temperature of
tumors as a function of the 808 nm laser (0.5 W/cm.sup.2)
irradiating time (laser irradiation was performed post 6 h
intravenous administration of TPA-T-TQ ONPs or saline for (a) and
(b)).
[0062] FIG. 12C depicts a graph showing tumor growth curves (of
different treatment groups of mice (** represents P<0.01, in
comparison between "ONPs+Laser" group and other treatment
groups).
[0063] FIG. 12D depicts a graph showing body weight changes of
different treatment groups of mice.
[0064] FIG. 13A shows histological H&E, fluorescence TUNEL and
PCNA staining of tumor slices at day 16 after treatment with ONPs
and laser, only ONPs, saline and laser, and only saline,
respectively.
[0065] FIG. 13B shows histological H&E staining for livers and
spleens on day 16 after the treatment with ONPs and laser, only
ONPs, saline and laser, and only saline, respectively.
[0066] FIG. 14A depicts graphs summarizing blood biochemistry data
of healthy Balb/c mice treated with TPA-T-TQ ONPs for 7 days.
[0067] FIG. 14B depicts graphs summarizing hematology data of
healthy Balb/c mice treated with TPA-T-TQ ONPs for 7 days (the
untreated mice were used as the control).
DETAILED DESCRIPTION
[0068] The following definitions are provided for the purpose of
understanding the present subject matter and for constructing the
appended patent claims.
DEFINITIONS
[0069] It should be understood that the drawings described above or
below are for illustration purposes only. The drawings are not
necessarily to scale, with emphasis generally being placed upon
illustrating the principles of the present teachings. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0070] Throughout the application, where compositions are described
as having, including, or comprising specific components, or where
processes are described as having, including, or comprising
specific process steps, it is contemplated that compositions of the
present teachings can also consist essentially of, or consist of,
the recited components, and that the processes of the present
teachings can also consist essentially of, or consist of, the
recited process steps.
[0071] In the application, where an element or component is said to
be included in and/or selected from a list of recited elements or
components, it should be understood that the element or component
can be any one of the recited elements or components, or the
element or component can be selected from a group consisting of two
or more of the recited elements or components. Further, it should
be understood that elements and/or features of a composition, an
apparatus, or a method described herein can be combined in a
variety of ways without departing from the spirit and scope of the
present teachings, whether explicit or implicit herein
[0072] The use of the terms "include," "includes", "including,"
"have," "has," or "having" should be generally understood as
open-ended and non-limiting unless specifically stated
otherwise.
[0073] The use of the singular herein includes the plural (and vice
versa) unless specifically stated otherwise. In addition, where the
use of the term "about" is before a quantitative value, the present
teachings also include the specific quantitative value itself,
unless specifically stated otherwise. As used herein, the term
"about" refers to a .+-.10% variation from the nominal value unless
otherwise indicated or inferred.
[0074] It should be understood that the order of steps or order for
performing certain actions is immaterial so long as the present
teachings remain operable. Moreover, two or more steps or actions
may be conducted simultaneously.
[0075] As used herein, "heteroaryl" refers to an aromatic
monocyclic ring system containing at least one ring heteroatom
selected from oxygen (O), nitrogen (N), sulfur (S), silicon (Si),
and selenium (Se) or a polycyclic ring system where at least one of
the rings present in the ring system is aromatic and contains at
least one ring heteroatom. Polycyclic heteroaryl groups include two
or more heteroaryl rings fused together and monocyclic heteroaryl
rings fused to one or more aromatic carbocyclic rings, non-aromatic
carbocyclic rings, and/or non-aromatic cycloheteroalkyl rings. A
heteroaryl group, as a whole, can have, for example, 5 to 22 ring
atoms and contain 1-5 ring heteroatoms (i.e., 5-20 membered
heteroaryl group). The heteroaryl group can be attached to the
defined chemical structure at any heteroatom or carbon atom that
results in a stable structure. Generally, heteroaryl rings do not
contain O--O, S--S, or S--O bonds. However, one or more N or S
atoms in a heteroaryl group can be oxidized (e.g., pyridine
N-oxide, thiophene S-oxide, thiophene S,S-dioxide). Examples of
heteroaryl groups include, for example, the 5- or 6-membered
monocyclic and 5-6 bicyclic ring systems shown below:
##STR00011##
where T is O, S, NH, N-alkyl, N-aryl, N-(arylalkyl) (e.g.,
N-benzyl), SiH.sub.2, SiH(alkyl), Si(alkyl).sub.2, SiH(arylalkyl),
Si(arylalkyl).sub.2, or Si(alkyl)(arylalkyl). Examples of such
heteroaryl rings include pyrrolyl, furyl, thienyl, pyridyl,
pyrimidyl, pyridazinyl, pyrazinyl, triazolyl, tetrazolyl,
pyrazolyl, imidazolyl, isothiazolyl, thiazolyl, thiadiazolyl,
isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl, benzofuryl,
benzothienyl, quinolyl, 2-methylquinolyl, isoquinolyl, quinox-alyl,
quinazolyl, benzotriazolyl, benzimidazolyl, benzothiazolyl,
benzisothiazolyl, benzisoxazolyl, benzoxadiazolyl, benzoxazolyl,
cinnolinyl, 1H-indazolyl, 2H-indazolyl, indolizinyl, isobenzofuyl,
naphthyridinyl, phthalazinyl, pteridinyl, purinyl,
oxazolopyridinyl, thiazolopyridinyl, imidazopyridinyl,
furopyridinyl, thienopyridinyl, pyridopyrimidinyl, pyridopyrazinyl,
pyridopyridazinyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl
groups, and the like. Further examples of heteroaryl groups include
4,5,6,7-tetrahydroindolyl, tetrahydroquinolinyl,
benzothienopyridinyl, benzofuropyridinyl groups, and the like. In
some embodiments, heteroaryl groups can be substituted as described
herein.
[0076] As used herein, "halo" or "halogen" refers to fluoro,
chloro, bromo, and iodo. As used herein, "alkyl" refers to a
straight-chain or branched saturated hydrocarbon group. Examples of
alkyl groups include methyl (Me), ethyl (Et), propyl (e.g.,
n-propyl and z'-propyl), butyl (e.g., n-butyl, z'-butyl, sec-butyl,
tert-butyl), pentyl groups (e.g., n-pentyl, z'-pentyl, -pentyl),
hexyl groups, and the like. In various embodiments, an alkyl group
can have 1 to 40 carbon atoms (i.e., C1-40 alkyl group), for
example, 1-30 carbon atoms (i.e., C1-30 alkyl group). In some
embodiments, an alkyl group can have 1 to 6 carbon atoms, and can
be referred to as a "lower alkyl group." Examples of lower alkyl
groups include methyl, ethyl, propyl (e.g., n-propyl and
z'-propyl), and butyl groups (e.g., n-butyl, z'-butyl, sec-butyl,
ten-butyl). In some embodiments, alkyl groups can be substituted as
described herein. An alkyl group is generally not substituted with
another alkyl group, an alkenyl group, or an alkynyl group.
[0077] As used herein, "alkenyl" refers to a straight-chain or
branched alkyl group having one or more carbon-carbon double bonds.
Examples of alkenyl groups include ethenyl, propenyl, butenyl,
pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl groups, and
the like. The one or more carbon-carbon double bonds can be
internal (such as in 2-butene) or terminal (such as in 1-butene).
In various embodiments, an alkenyl group can have 2 to 40 carbon
atoms (i.e., C2-40 alkenyl group), for example, 2 to 20 carbon
atoms (i.e., C2-20 alkenyl group). In some embodiments, alkenyl
groups can be substituted as described herein. An alkenyl group is
generally not substituted with another alkenyl group, an alkyl
group, or an alkynyl group.
[0078] As used herein, a "fused ring" or a "fused ring moiety"
refers to a polycyclic ring system having at least two rings where
at least one of the rings is aromatic and such aromatic ring
(carbocyclic or heterocyclic) has a bond in common with at least
one other ring that can be aromatic or non-aromatic, and
carbocyclic or heterocyclic. These polycyclic ring systems can be
highly p-conjugated and optionally substituted as described
herein.
[0079] As used herein, "heteroatom" refers to an atom of any
element other than carbon or hydrogen and includes, for example,
nitrogen, oxygen, silicon, sulfur, phosphorus, and selenium.
[0080] As used herein, "aryl" refers to an aromatic monocyclic
hydrocarbon ring system or a polycyclic ring system in which two or
more aromatic hydrocarbon rings are fused (i.e., having a bond in
common with) together or at least one aromatic monocyclic
hydrocarbon ring is fused to one or more cycloalkyl and/or
cycloheteroalkyl rings. An aryl group can have 6 to 24 carbon atoms
in its ring system (e.g., C6-24 aryl group), which can include
multiple fused rings. In some embodiments, a polycyclic aryl group
can have 8 to 24 carbon atoms. Any suitable ring position of the
aryl group can be covalently linked to the defined chemical
structure. Examples of aryl groups having only aromatic carbocyclic
ring(s) include phenyl, 1-naphthyl (bicyclic), 2-naphthyl
(bicyclic), anthracenyl (tricyclic), phenanthrenyl (tricyclic),
pentacenyl (pentacyclic), and like groups. Examples of polycyclic
ring systems in which at least one aromatic carbocyclic ring is
fused to one or more cycloalkyl and/or cycloheteroalkyl rings
include, among others, benzo derivatives of cyclopentane (i.e., an
indanyl group, which is a 5,6-bicyclic cycloalkyl/aromatic ring
system), cyclohexane (i.e., a tetrahydronaphthyl group, which is a
6,6-bicyclic cycloalkyl/aromatic ring system), imidazoline (i.e., a
benzimidazolinyl group, which is a 5,6-bicyclic
cycloheteroalkyl/aromatic ring system), and pyran (i.e., a
chromenyl group, which is a 6,6-bicyclic cycloheteroalkyl/aromatic
ring system). Other examples of aryl groups include benzodioxanyl,
benzodioxolyl, chromanyl, indolinyl groups, and the like. In some
embodiments, aryl groups can be substituted as described herein. In
some embodiments, an aryl group can have one or more halogen
substituents, and can be referred to as a "haloaryl" group.
Perhaloaryl groups, i.e., aryl groups where all of the hydrogen
atoms are replaced with halogen atoms (e.g., --C6F5), are included
within the definition of "haloaryl." In certain embodiments, an
aryl group is substituted with another aryl group and can be
referred to as a biaryl group. Each of the aryl groups in the
biaryl group can be substituted as disclosed herein.
[0081] As used herein, a "donor" material refers to an organic
material, for example, an organic nanoparticle material, having
holes as the majority current or charge carriers.
[0082] As used herein, an "acceptor" material refers to an organic
material, for example, an organic nanoparticle material, having
electrons as the majority current or charge carriers.
[0083] As used herein, a "theranostic agent" refers to an organic
material, for example, an organic nanoparticle material, having
both diagnostic and therapeutic capabilities.
[0084] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which the presently described subject
matter pertains.
[0085] Where a range of values is provided, for example,
concentration ranges, percentage ranges, or ratio ranges, it is
understood that each intervening value, to the tenth of the unit of
the lower limit, unless the context clearly dictates otherwise,
between the upper and lower limit of that range and any other
stated or intervening value in that stated range, is encompassed
within the described subject matter. The upper and lower limits of
these smaller ranges may independently be included in the smaller
ranges, and such embodiments are also encompassed within the
described subject matter, subject to any specifically excluded
limit in the stated range. Where the stated range includes one or
both of the limits, ranges excluding either or both of those
included limits are also included in the described subject
matter.
[0086] Throughout the application, descriptions of various
embodiments use "comprising" language. However, it will be
understood by one of skill in the art, that in some specific
instances, an embodiment can alternatively be described using the
language "consisting essentially of" or "consisting of".
[0087] For purposes of better understanding the present teachings
and in no way limiting the scope of the teachings, unless otherwise
indicated, all numbers expressing quantities, percentages or
proportions, and other numerical values used in the specification
and claims, are to be understood as being modified in all instances
by the term "about". Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the following specification
and attached claims are approximations that may vary depending upon
the desired properties sought to be obtained. At the very least,
each numerical parameter should at least be construed in light of
the number of reported significant digits and by applying ordinary
rounding techniques.
Theranostic Agents
[0088] The present subject matter contemplates theranostic agents,
or agents useful for both diagnostic and therapeutic purposes. A
theranostic agent, as contemplated herein, can include at least one
small molecule organic compound with absorption in the
near-infrared (NIR) interrogation window (700-900 nm). The compound
can be an organic nanoparticle (ONP). The theranostic agents
described herein can provide ideal contrast agents for light
triggered diagnostic/therapeutic techniques, such as photoacoustic
imaging (PAI) associated with photothermal therapy (PTT). The
theranostic agents described herein demonstrate excellent thermal
and photothermal stabilities, as well as significant resistance to
photobleaching and RONS. The theranostic agents described herein
further exhibit excellent photothermal conversion performance when
exposed to NIR light.
[0089] In an embodiment, the present theranostic agent is a small
molecule, organic compound that has:
[0090] a donor unit selected from the group consisting of
##STR00012## ##STR00013## ##STR00014##
and
[0091] an acceptor unit (A) selected from the group consisting
of:
##STR00015##
[0092] wherein the compound is arranged in a form selected from the
group consisting of D-A, D-A-D, A-D-A, D-D-A-D-D, A-A-D-A-A,
D-A-D-A-D, A-D-A-D-A,
[0093] wherein D and D' represent the donor unit;
[0094] wherein A represents the acceptor unit;
[0095] wherein each of X and X' is selected from the group
consisting of O, S, Se, and Te;
[0096] wherein each of R, R', R'' R''', or R'''' is unsubstituted
or substituted and is selected from the group consisting of F, H,
alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group,
sulfonic group, alkylthio, and alkoxy group; and
[0097] wherein at least one of R, R', R'' R''', or R'''' is a
terminal functional group having a substituent independently
selected from the group consisting of N.sub.3, NCS, SH, NH.sub.2,
COOH, alkyne, N-hydroxysuccinimide ester, maleimide, hydrazide,
nitrone group, --CHO, --OH, halide, and a charged ionic group;
and
[0098] wherein at least one of R, R', R'' R''', and R'''' is other
than H.
[0099] In a further embodiment, the compound has one of the
following structural formulae:
##STR00016##
[0100] wherein each of X and X' is selected from the group
consisting of O, S, Se, and Te;
[0101] wherein each of D and D' is selected from the group
consisting of
##STR00017## ##STR00018##
[0102] wherein each of R, R', R'', and R''' is unsubstituted or
substituted, and is selected from the group consisting of F, H,
alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group,
sulfonic group, alkylthio, and alkoxy group;
[0103] wherein at least one of R, R', R'', and R''' is a terminal
functional group having a substituent independently selected from
the group consisting of N.sub.3, NCS, SH, NH.sub.2, COOH, alkyne,
N-hydroxysuccinimide ester, maleimide, hydrazide, nitrone group,
--CHO, --OH, halide, and a charged ionic group; and
[0104] wherein at least one of R, R', R'', and R''' is other than
H.
[0105] In an embodiment, the compound has the following structural
formula:
##STR00019##
[0106] wherein X is selected from the group consisting of O, S, Se,
and Te;
[0107] wherein each of R, R' R'', R''', R'''' R''''', R'''''', and
R''''''' is unsubstituted or substituted, and is selected from the
group consisting of F, H, alkyl, unsaturated alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group,
amino group, sulfonic group, alkylthio, and alkoxy group;
[0108] wherein at least one of R, R', R'' R''', R'''', R''''',
R'''''', and R''''''' is a terminal functional group having a
substituent independently selected from the group consisting of
N.sub.3, NCS, SH, NH.sub.2, COOH, alkyne, N-Hydroxysuccinimide
ester, maleimide, hydrazide, nitrone group, --CHO, --OH, halide,
and a charged ionic group; and
[0109] wherein at least one of R, R' R'', R''', R'''' R''''',
R'''''', and R''''''' is other than H.
[0110] In an embodiment, the compound has the following structural
formula:
##STR00020##
[0111] wherein each of R, R' R'', R''', R'''', R''''', R'''''', and
R''''''' is unsubstituted or substituted, and is selected from the
group consisting of F, H, alkyl, unsaturated alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group,
amino group, sulfonic group, alkylthio, and alkoxy group;
[0112] wherein at least one of R, R', R'', R''', R'''', R''''',
R'''''', and R''''''' is a terminal functional group having a
substituent independently selected from the group consisting of
N.sub.3, NCS, SH, NH.sub.2, COOH, alkyne, N-Hydroxysuccinimide
ester, maleimide, hydrazide, nitrone group, --CHO, --OH, halide,
and a charged ionic group; and
wherein at least one of R, R', R'' R''', R'''', R''''', R'''''',
and R''''''' is other than H
[0113] In an embodiment, the compound is:
##STR00021##
[0114] An exemplary reaction scheme for preparing the TPA-T-TQ
compound is provided below:
##STR00022## ##STR00023##
[0115] In an embodiment the compounds are provided as
nanoparticles. The nanoparticles can be synthesized using an
amphiphilic matrix as shown in the reaction scheme depicted in FIG.
8A and described in detail herein.
Identifying Tumor and Stopping or Inhibiting Tumor Growth
[0116] The present compounds can be administered to a patient as a
contrast agent for locating a tumor site in the patient using in
vivo imaging techniques, e.g., photoacoustic imaging. The compounds
can be administered by intravenous injection, for example. As set
forth in detail herein, in vivo imaging studies demonstrate that
the compounds can serve as an effective probe for PAI in a
high-contrast manner. Once the tumor site has been determined, the
tumor site can be irradiated with near-infrared light which, when
combined with the present compounds, can stop or inhibit the growth
of the tumor. In an embodiment, the compounds can be administered
to the patient six hours prior to PA imaging and PTT treatment of
the tumor.
[0117] In vivo tumor growth kinetics with a xenograft 4T1
tumor-bearing mouse model reveal that PTT using the present
compounds effectively suppresses and stops tumor growth. The
present compounds demonstrate rapid temperature elevation in a
tumor site under NIR light irradiation, which results in
heat-caused tumor inhibition. As described herein, this superior
antitumor efficacy is also confirmed by histological and
immune-histochemical staining of tumor slices. Various functional
and targeted groups can be introduced to the compounds to
facilitate specific targeting of a desired biological species.
According to an embodiment, one or more peptides can be conjugated
to the present compounds.
[0118] As the present compounds are completely organic, these
compounds show good biocompatibility, and no detectable side
toxicity based on histological examinations and blood tests. The
compounds demonstrate ultra-high stability and good
photothermal/photoacoustic performance, making them promising
candidates for in vivo diagnosis and therapy applications.
[0119] The present teachings are illustrated by the following
examples.
EXAMPLES
Materials and Instruments
[0120] .sup.1H (400 MHz) and .sup.13C (100 MHz) nuclear magnetic
resonance (NMR) spectra were recorded on a Bruker AV 400
spectrometer by using CDCl.sub.3 or DMSO-d.sub.6 as the solvent.
High-resolution mass spectra (HRMS) were measured on a GCT premier
CAB048 mass spectrometer in MALDI-TOF mode. Thermogravimetric
analysis (TGA) measurement was conducted on a TA TGA Q5000 with a
heating rate of 10.degree. C./min under nitrogen atmosphere. The
UV-vis-NIR absorption spectra were performed on a PerkinElmer
Lambda 365 spectrophotometer. The photoluninescence (PL) spectra
were conducted using a Horiba Fluorolog-3 spectrofluorometer.
Dynamic light scattering (DLS) was performed on a 90 plus particle
size analyzer. Transmission electron microscopy (TEM) images were
obtained on a JEM-2010F transmission electron microscope with an
accelerating voltage of 200 kV.
[0121] Quantitative data were expressed as mean.+-.standard
deviation (SD). Statistical comparisons were made by ANOVA analysis
and two-sample Student's t-test. P value <0.05 was considered
statistically significant.
Example 1
Synthesis of TPA-T-TQ
4-(tert-Butyl)-N-(p-tolyl)aniline (3)
[0122] 4-(tert-Butyl)aniline (1.94 g, 13 mmol),
1-bromo-4-methylbenzene (1.71 g, 10 mmol), sodium tert-butoxide
(1.25 g, 13 mmol), and palladium (II) acetate (Pd(OAc)2) (45 mg,
0.2 mmol) were added into a 100 mL two-necked round-bottom flask.
The flask was vacuumed and purged with dry nitrogen three times.
Then tri-tert-butylphosphine (P(.sup.tBu)3, 0.25 mmol, 1 M toluene
solution, 0.25 mL) and anhydrous toluene (50 mL) were added, and
the resulting mixture was heated to reflux and stirred for 24 h in
the absence of light. After cooling down to room temperature, water
was added, and the mixture was extracted with dichloromethane. The
organic phase was combined, and dried with MgSO.sub.4. After the
removal of the solvent under reduced pressure, the residue was
purified by column chromatography on silica gel using
dichloromethane/hexane (v/v 1:4) as the eluent to result in
4-(tert-butyl)-N-(p-tolyl)aniline as a colorless solid (75% yield).
.sup.1H NMR (400 MHz, CDCl.sub.3, 25.degree. C.) .delta. (ppm):
7.29 (d, 2H), 7.09 (d, 2H), 7.03-6.97 (m, 4H), 5.56 (br, 1H), 2.32
(s, 3H), 1.33 (s, 9H). .sup.13C NMR (100 MHz, CDCl.sub.3,
25.degree. C.) .delta. (ppm): 143.48, 141.20, 140.93, 130.29,
129.83, 126.10, 118.19, 117.16, 34.14, 31.51, 20.67.
4-Bromo-N-(4-(tert-butyl)phenyl)-N-(p-tolyl)aniline (5)
[0123] 4-(tert-Butyl)-N-(p-tolyl)aniline (1.68 g, 7 mmol),
1-bromo-4-iodobenzene (1.98 g, 7 mmol), 1,10-phenanthrothline (0.27
g, 1.5 mmol), copper (I) chloride (0.15 g, 1.5 mmol), and potassium
hydroxide (1.68 g, 30 mmol) were added into a 100 mL two-necked
round-bottom flask. The flask was vacuumed and purged with dry
nitrogen three times. Then anhydrous toluene (50 mL) was added, and
the resulting mixture was heated to reflux and stirred for 24 h.
After cooling down to room temperature, water was added, and the
mixture was extracted with dichloromethane. The organic phase was
combined, and dried with MgSO.sub.4. After the removal of the
solvent under reduced pressure, the residue was purified by column
chromatography on silica gel using dichloromethane/hexane (v/v 1:6)
as the eluent to result in
4-bromo-N-(4-(tert-butyl)phenyl)-N-(p-tolyl)aniline as a white
solid (73% yield). .sup.1H NMR (400 MHz, CDCl.sub.3, 25.degree. C.)
.delta. (ppm): 7.22 (d, 2H), 7.09-6.95 (m, 8H), 6.93 (s, 2H), 2.30
(s, 3H), 1.30 (s, 9H). .sup.13C NMR (100 MHz, CDCl.sub.3,
25.degree. C.) .delta. (ppm): 145.52, 144.75, 142.74, 131.91,
130.01, 129.76, 126.14, 125.88, 124.88, 124.07, 123.77, 122.74,
34.21, 31.46, 20.80.
Thiophen-2-ylboronic acid (6)
[0124] Into a 100 mL two-necked round-bottom flask, thiophene (1.68
g, 20 mmol) and anhydrous THF (40 mL) were added. The flask was
then vacuumed and purged with dry nitrogen three times. Then the
mixture was cooled with dry ice-acetone to -78.degree. C., and
maintained for 15 min, followed by the addition of n-butyllithium
(.sup.nBuLi, 2.5 M hexane solution, 8.8 mL, 22 mmol). The reaction
mixture was stirred at -78.degree. C. for 30 min before slowly
warmed to -20.degree. C., and stirred for another 30 min.
Afterwards, the mixture was cooled to -78.degree. C., and
trisethylborate (2.5 mL, 22 mmol) was added. The mixture was
stirred at -78.degree. C. for another 1 h, and then slowly warmed
to room temperature, and stirred overnight. The reaction mixture
was then treated with aqueous HCl (1 M), and extracted with
dichloromethane three times. The organic phase was combined, and
dried with MgSO.sub.4. After the removal of the solvent under
reduced pressure, the product was further purified by
recrystallization to give thiophen-2-ylboronic acid as a white
solid (65% yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6, 25.degree.
C.) .delta. (ppm): 8.20 (d, 2H), 7.74 (dd, 1H), 7.67 (dd, 1H), 7.17
(dd, 1H). .sup.13C NMR (100 MHz, DMSO-d.sub.6, 25.degree. C.)
.delta. (ppm): 136.41, 132.02, 128.54.
4-(tert-Butyl)-N-(4-(thiophen-2-yl)phenyl)-N-(p-tolyl)aniline
(7)
[0125] 4-Bromo-N-(4-(tert-butyl)phenyl)-N-(p-tolyl)aniline (1.97 g,
5 mmol), thiophen-2-ylboronic acid (0.64 g, 5 mmol),
Pd(PPh.sub.3).sub.4 (115 mg, 0.1 mmol), and K.sub.2CO.sub.3 (2.76
g, 20 mmol) were added into a 100 mL two-necked round-bottom flask.
The flask was vacuumed and purged with dry nitrogen three times.
Then anhydrous THF (40 mL) and water (10 mL) were added, and the
mixture was heated to reflux and stirred for 24 h in the absence of
light. After cooling down to room temperature, water was added, and
the mixture was extracted with dichloromethane. The organic phase
was combined, and dried with MgSO.sub.4. After the removal of the
solvent under reduced pressure, the residue was purified by column
chromatography on silica gel using dichloromethane/hexane (v/v 1:5)
as the eluent to result in
4-(tert-butyl)-N-(4-(thiophen-2-yl)phenyl)-N-(p-tolyl)aniline as a
light yellow solid (81% yield). .sup.1H NMR (400 MHz, CDCl.sub.3,
25.degree. C.) .delta. (ppm): 7.46-7.42 (m, 2H), 7.25-7.23 (m, 2H),
7.19 (d, 2H), 7.10-7.00 (m, 9H), 2.32 (s, 3H), 1.31 (s, 9H).
.sup.13C NMR (100 MHz, CDCl.sub.3, 25.degree. C.) .delta. (ppm):
147.65, 145.80, 145.03, 144.85, 144.54, 132.84, 129.96, 127.95,
127.71, 126.63, 126.09, 124.96, 123.84, 123.76, 122.75, 121.98,
34.31, 31.46, 20.87.
4-(tert-Butyl)-N-(p-tolyl)-N-(4-(5-(tributylstannyl)thiophen-2-yl)phenyl)a-
niline (8)
[0126]
4-(tert-Butyl)-N-(4-(thiophen-2-yl)phenyl)-N-(p-tolyl)aniline (1.2
g, 3 mmol) was added into a 100 mL two-necked round-bottom flask.
The flask was vacuumed and purged with dry nitrogen three times.
Then anhydrous THF (50 mL) was added, and the resulting mixture was
cooled with dry ice-acetone to -78.degree. C., and maintained for
15 min, followed by the addition of n-butyllithium (.sup.nBuLi, 2.5
M hexane solution, 1.25 mL, 3.2 mmol). The mixture was stirred at
-78.degree. C. for 2 h. Afterwards, tri-n-butyltin chloride (0.9
mL, 3.3 mmol) was added, and the mixture was slowly warmed to room
temperature, and stirred overnight. Water was added to quench the
reaction, and the mixture was extracted with dichloromethane three
times. The organic phase was combined, and dried with MgSO.sub.4.
After the removal of the solvent under reduced pressure,
4-(tert-butyl)-N-(p-tolyl)-N-(4-(5-(tributylstannyl)thiophen-2-yl)phenyl)-
aniline was obtained as a brownish oil, and it was used without
further purification.
4,7-Dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole (9)
[0127] The mixture of 4,7-dibromobenzo[c][1,2,5]thiadiazole (8.82
g, 30 mmol), sulfuric acid (60 mL), fuming sulfuric acid (20 mL),
fuming nitric acid (50 mL) was stirred at 0.degree. C. for 4 h to
finish the nitration reaction. Then the mixture was poured into ice
water (500 mL) slowly to get a suspension, and filtered through a
Buchner funnel, washed with water several times, and dried in
vacuum to get 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole as
a light yellow powder (88% yield). .sup.13C NMR (100 MHz,
DMSO-d.sub.6, 25.degree. C.) .delta. (ppm): 151.98, 144.13,
111.84.
4,4'-((5,6-Dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(thiophene-5,2-di-
yl))bis(N-(4-(tert-butyl)phenyl)-N-(p-tolyl)aniline) (10)
[0128]
4-(tert-Butyl)-N-(p-tolyl)-N-(4-(5-(tributylstannyl)thiophen-2-yl)p-
henyl)aniline (2.06 g, 3 mmol),
4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole (0.46 g, 1.2
mmol), and Pd(PPh.sub.3).sub.4 (58 mg, 0.05 mmol) were added into a
100 mL two-necked round-bottom flask. The flask was vacuumed and
purged with dry nitrogen three times. Then anhydrous THF (40 mL)
was added, and the mixture was heated to reflux and stirred for 24
h in the absence of light. After cooling down to room temperature,
water was added, and the mixture was extracted with
dichloromethane. The organic phase was combined, and dried with
MgSO.sub.4. After the removal of the solvent under reduced
pressure, the crude product was purified by column chromatography
on silica gel using dichloromethane/hexane (v/v 1:3) as the eluent
to result in
4,4'-((5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(thiophene-5,2-d-
iyl))bis(N-(4-(tert- butyl)phenyl)-N-(p-tolyl)aniline) as a dark
blue solid (79% yield). The .sup.1H NMR spectrum of compound 10 is
depicted in FIG. 1. The .sup.13C NMR spectrum of compound 10 is
depicted in FIG. 2. The HRMS of compound 10 is depicted in FIG. 3.
.sup.1H NMR (400 MHz, CDCl.sub.3, 25.degree. C.) .delta. (ppm):
7.49 (m, 6H), 7.28 (m, 6H), 7.11 (m, 4H), 7.05 (m, 12H), 2.34 (s,
6H), 1.33 (m, 18H). .sup.13C NMR (100 MHz, CDCl.sub.3, 25.degree.
C.) .delta. (ppm): 152.02, 151.40, 148.86, 146.44, 144.61, 144.43,
141.20, 133.44, 132.18, 130.06, 127.53, 126.88, 126.21, 125.73,
125.38, 124.38, 122.80, 121.78, 120.28, 34.37, 31.44, 20.91. HRMS
(MALDI-TOF, m/z): [M].sup.+ calcd for
C.sub.60H.sub.52N.sub.6O.sub.4S.sub.3, 1016.3212; found,
1016.3249.
4,7-Bis(5-(4-((4-(tert-butyl)phenyl)(p-tolyl)amino)phenyl)thiophen-2-yl)be-
nzo[c][1,2,5]thiadiazole-5,6-diamine (11)
[0129]
4,4'-((5,6-Dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(thiophene-
-5,2-diyl))bis(N-(4-(tert-butyl)phenyl)-N-(p-tolyl)aniline) (0.92
g, 0.9 mmol), iron powder (1.1 g, 20 mmol) and acetic acid (50 mL)
were suspended in a 100 mL round-bottom flask, and the mixture was
heated to 80.degree. C., and stirred for 4 h. After cooling down to
room temperature, water was added, and the mixture was extracted
with dichloromethane. The organic layer was washed with water and
NaHCO.sub.3 aqueous solution. The organic phase was combined, and
dried with MgSO.sub.4. After the removal of the solvent under
reduced pressure,
4,7-bis(5-(4-((4-(tert-butyl)phenyl)(p-tolyl)amino)phenyl)thiophen-2-yl)b-
enzo[c][1,2,5]thiadiazole-5,6-diamine was obtained as a dark red
solid and used without further purification.
4,4'-((6,7-Diphenyl-[1,2,5]thiadiazolo[3,4-g]quinoxaline-4,9-diyl)bis(thio-
phene-5,2-diyl))bis(N-(4-(tert-butyl)phenyl)-N-(p-tolyl)aniline)
(TPA-T-TQ)
[0130]
4,7-bis(5-(4-((4-(tert-butyl)phenyl)(p-tolyl)amino)phenyl)thiophen--
2-yl)benzo[c][1,2,5]thiadiazole-5,6-diamine (0.86 g, 0.9 mmol) was
dissolved in the mixture of chloroform (20 mL) and acetic acid (20
mL) in a 100 mL round-bottom flask, and benzil (0.32 g, 1.5 mmol)
was added. Then the mixture was heated to reflux and stirred for 12
h. After cooling down to room temperature, water was added, and the
mixture was extracted with dichloromethane. The organic layer was
washed with water and NaHCO.sub.3 aqueous solution. The organic
phase was combined, and dried with MgSO.sub.4. After the removal of
the solvent under reduced pressure, the crude product was purified
by column chromatography on silica gel using dichloromethane/hexane
(v/v 1:3) as the eluent to result in
4,4'-((6,7-diphenyl-[1,2,5]thiadiazolo[3,4-g]quinoxaline-4,9-diyl)bis(thi-
ophene-5,2-diyl))bis(N-(4-(tert- butyl)phenyl)-N-(p-tolyl)aniline)
as a yellow-green solid (76% yield). The .sup.1H NMR spectrum of
TPA-T-TQ is depicted in FIG. 4. The .sup.13C NMR spectrum of
TPA-T-TQ is depicted in FIG. 5. The HRMS of TPA-T-TQ is depicted in
FIG. 6. .sup.1H NMR (400 MHz, CDCl.sub.3, 25.degree. C.) .delta.
(ppm): 8.94 (br, 2H), 7.77 (m, 4H), 7.55 (br, 4H), 7.45-7.32 (m,
8H), 7.29 (d, 4H), 7.15-7.01 (m, 16H), 2.35 (s, 6H), 1.34 (s, 18H).
.sup.13C NMR (100 MHz, CDCl.sub.3, 25.degree. C.) .delta. (ppm):
152.39, 151.66, 147.86, 145.95, 144.97, 144.80, 138.08, 134.90,
134.47, 132.98, 130.88, 130.00, 129.50, 128.94, 128.46, 128.07,
126.44, 126.14, 125.14, 124.04, 122.56, 122.19, 122.80, 120.50,
34.35, 31.48, 20.91. HRMS (MALDI-TOF, m/z): [M].sup.+ calcd for
C.sub.74H.sub.62N.sub.6S.sub.3, 1130.4198; found, 1130.4208.
Example 2
Synthesis of TPA-T-TQ Organic Nanoparticles (ONPs)
[0131] 1 mL of tetrahydrofuran (THF) solution containing 1 mg of
TPA-T-TQ compound, and 2 mg of
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000 (DSPE-PEG.sub.2000) was poured into 10 mL of deionized
water. The mixture was sonicated for 2 min using a microtip probe
sonicator at 12 W output (XL2000, Misonix Incorporated, NY). The
residue THF solvent was evaporated by violent stirring of the
suspension at room temperature in fume hood overnight, and a
colloidal solution was obtained and directly used. FIG. 8A depicts
a schematic for preparing the organic small molecular nanoparticles
using the nanoprecipitation method. FIG. 8B depicts the UV-vis-NIR
absorption spectra of TPA-T-TQ in THF solution and the encapsulated
ONPs in water. The inset shows the photographs of (i) the TPA-T-TQ
THF solution, and (ii) the as-prepared ONPs in water. FIG. 8C
depicts the photoluminescence (PL) spectra of TPA-T-TQ in THF
solution and the encapsulated ONPs in water. FIG. 8D depicts the
TEM image of the TPA-T-TQ ONPs. FIG. 8E depicts the DLS profile of
the TPA-T-TQ ONPs. The nanoparticles were negatively stained with
uranyl acetate.
Example 3
Photo- and RONS-Stability Studies
[0132] For the photostability study, PBS solutions (pH 7.4) of
TPA-T-TQ ONPs and ICG were irradiated under 808-nm laser (0.8
W/cm.sup.2), and the absorption spectra were measured at different
time points. For the anti-photobleaching study, the temperatures of
the sample solutions were recorded during five circles of heating
and cooling. In one heating-cooling circle, the NIR laser first
irradiated the samples for 5 min to reach a steady state, then the
laser was removed and the samples were naturally cooled down to
ambient temperature in 6 min.
[0133] For the RONS-stability study, two kinds of RONS were used,
i.e., ONOO.sup.- and *OH, which were prepared according to the
literature. The absorption and photoluminescence spectra were
recorded before and after addition of 0.4 mM of RONS. The
stabilities of the contrast agents in terms of thermal and
photothermal stabilities as well as photobleaching and RONS
resistances are crucial for PAI/PTT applications in vivo because
incorrect or misleading signals, weakened therapeutic efficacy, and
harmful side effects can result if the structure of the agent is
destroyed, especially in living systems. Thermogravimetric analysis
(TGA) was first carried out to measure the thermal stability of
TPA-T-TQ. The decomposition temperature for 5% weight loss of
TPA-T-TQ was above 400.degree. C. (FIG. 7), suggesting excellent
thermal stability. Then the photostability of TPA-T-TQ ONPs was
evaluated along with ICG under continuous 808 nm laser irradiation
(0.8 W/cm.sup.2) by recording the apparent colors and absorption
spectra after laser irradiation for different times. FIG. 9A
depicts IR thermal images of the ONPs and ICG in PBS solutions (100
.mu.M) under 808-nm laser irradiation (0.8 W/cm.sup.2) for
different times. FIG. 9B depicts a graph showing photothermal
conversion behavior of TPA-T-TQ ONPs at different concentrations
(5-100 .mu.M) under 808 nm light irradiation at a power intensity
of 0.8 W/cm.sup.2. FIG. 9C depicts a graph comparing the
photothermal conversion behavior of TPA-T-TQ ONPs and ICG in PBS
solution at the same concentration (100 .mu.M) (the 808-nm light
(0.8 W/cm.sup.2) was irradiated for 5 min)
[0134] As shown in FIGS. 9D and 9E, the colors and absorption
spectra of TPA-T-TQ ONPs are nearly unchanged during 15 min NIR
light irradiation duration, whereas the blue-green color of ICG
solution gradually disappears, and the maximal absorption intensity
nearly drops to zero after being exposed to the NIR light for 15
min. Noteworthy is that the optical properties of TPA-T-TQ ONPs are
identical to the original state even after continuous laser
illumination (0.8 W/cm.sup.2) for an hour. Then, the
anti-photobleaching properties of TPA-T-TQ ONPs and ICG were
evaluated by alternative heating and cooling processes (the NIR
laser first irradiated the samples for 5 min to heat them up, then
the laser was removed, and the samples were naturally cooled down
to ambient temperature in 6 min). Interestingly, during five
circles of heating and cooling processes, the photothermal
conversion ability of the ONPs shows negligible change, while the
temperature elevation of ICG dramatically dropped to about 20%
(.DELTA.T.about.10.degree. C.) of the original value
(.DELTA.T.about.44.degree. C.) after two circles of heating-cooling
processes (FIG. 9F). These results provide solid confirmation that
TPA-T-TQ ONPs possess superior resistance to photobleaching
compared to the existent organic small molecules.
[0135] Another important issue related to in vivo applications is
the physiological stability against highly reactive molecules such
as RONS. RONS are a kind of signaling molecules which are necessary
to regulate physiological functions, but are overproduced as a
result of the presence of various diseases including inflammation,
cancers, and cardiovascular diseases. For cancer diagnosis and
treatment, it is considerably vital to use RONS-resistant agents to
get reliable imaging signal and therapy efficacy. Therefore, the
stability of TPA-T-TQ ONPs and ICG in the presence of two kinds of
RONS, peroxynitrite (ONOO.sup.-) and hydroxyl radical (*OH), were
measured under physiological conditions. The absorption spectra and
photographic solutions of TPA-T-TQ ONPs and ICG before and after
the treatment of RONS reagents are depicted in FIGS. 9G and 9H,
respectively. After the addition of ONOO.sup.- and *OH, the maximal
absorption intensity of ICG (at 780 nm) drops to about 60% and 4%
relative to the original value, respectively. In marked contrast,
there is nearly no change in the absorption spectra and solution
appearance of the ONPs after adding each RONS (FIG. 9I). FIG. 10
depicts the photoluminescence (PL) spectra of ICG in water before
and after the addition 0.4 mM of ONOO.sup.- and *OH for 1 min.
Example 4
Photothermal Performance
[0136] The PBS solution (pH 7.4) of TPA-T-TQ ONPs and ICG in
different concentrations were continuously exposed to NIR laser of
808 nm with a power intensity of 0.8 W/cm.sup.2 for 5 min. The
temperature was measured every 20 s and stopped until the
temperature nearly reached a plateau. The corresponding IR thermal
images were also recorded. To evaluate the photothermal conversion
property, we quantitatively measured the temperature change of
TPA-T-TQ ONP solution at different concentrations as a function of
808 nm laser (0.8 W/cm.sup.2) irradiation time. As depicted in FIG.
9B, temperature increases very fast initially, and reaches a
plateau after 3 min laser irradiation. It is noted that the
eventual temperature at the plateau depends on the ONP
concentration. The temperature increase of ONPs and ICG solutions
upon exposure to 808 nm laser irradiation was compared. As
displayed in FIG. 9C, the TPA-T-TQ ONPs (.DELTA.T.about.53.degree.
C. in 5 min) exhibit much higher plateau temperature and faster
temperature rise rate than ICG (.DELTA.T.about.43.degree. C. in 5
min), revealing superior photothermal conversion behavior of the
ONPs. The difference in temperature elevation of the two samples at
various irradiation times can be intuitively visualized from the
infrared (IR) thermal images (FIG. 9A).
Example 5
Animal Model
[0137] To establish the xenograft 4T1 tumor-bearing mouse model,
murine 4T1 breast cancer cells (1.times.10.sup.6) suspended in 50
.mu.L of RPMI-1640 medium were subcutaneously injected into the
right axillary space of each mouse. After about 7 days, mice with
tumor volumes at about 80-120 mm.sup.3 were used.
Example 6
In Vivo Photoacoustic Imaging
[0138] PA images were acquired at 770 nm at designated time
intervals post ONPs injection. The in vivo PAI by intravenous
injection of the TPA-T-TQ ONPs into xenograft 4T1 tumor-bearing
nude mice was investigated. Before ONP administration (0 h), there
is a weak PA signal at 770 nm probably attributed to the absorption
of endogenous melanin and hemoglobin in the NIR spectral region.
FIG. 11A depicts the PA spectrum of TPA-T-TQ ONPs in PBS solution
(110 .mu.g/mL based on TPA-T-TQ). FIG. 11B depicts PA amplitudes of
TPA-T-TQ ONPs at 770 nm as a function of TPA-T-TQ
concentration.
[0139] Compared with the PA image of the tumor at 0 h, the PA
brightness of the tumor site after intravenous injection of
TPA-T-TQ ONPs significantly increases over time, which reaches a
maximum at 6 h post-injection (FIG. 11C), indicating that 6 h
post-injection is the optimized time point for PA imaging and PTT
treatment of tumor. The time-dependent PA images of tumors are
presented in FIG. 11D. The PA signal at 6 h is 2.4-fold higher as
compared to that of the tumor background, indicating the prominent
enhanced permeability and retention (EPR) effect of the ONPs, which
leads to their efficient accumulation in the tumor tissue.
Example 7
In Vivo Photothermal Therapy
[0140] The xenograft 4T1 tumor-bearing mice were randomly divided
into 4 groups (n=6 per group), which were named "Only Saline",
"Saline+Laser", "Only ONPs", and "ONPs+Laser", respectively. For
"Only Saline" and "Only ONPs" groups, saline and TPA-T-TQ ONPs (250
.mu.g/mL based on TPA-T-TQ) were injected into 4T1 tumor-bearing
mice via the tail vein, respectively, without subsequent laser
irradiation. For "Saline+Laser" and "ONPs+Laser" groups, after
intravenous injection of saline and TPA-T-TQ ONPs (250 .mu.g/mL
based on TPA-T-TQ) for 6 h, respectively, the tumors of mice in
each group were continuously irradiated with 808 nm laser (0.5
mW/cm.sup.2) for 5 min. After a variety of treatments, the tumor
volumes and mouse body weights were measured every other day for 16
days. The tumor volume was calculated by the following equation:
Volume=Width.sup.2.times.Length/2.
[0141] The PTT capability of the ONPs was validated with the
xenograft 4T1 tumor mouse model. Tumor-bearing mice were randomly
assigned to 4 groups, which were named "Only Saline",
"Saline+Laser", "Only ONPs", and "ONPs+Laser", respectively. For
"Only Saline" and "Only ONPs" groups, Saline and TPA-T-TQ ONPs (250
.mu.g/mL based on TPA-T-TQ) were injected into 4T1 tumor-bearing
mice via the tail vein, respectively, without subsequent laser
irradiation. For "Saline+Laser" and "ONPs+Laser" groups, after
intravenous injection of saline and TPA-T-TQ ONPs (250 .mu.g/mL
based on TPA-T-TQ) for 6 h, respectively, the tumors of mice in
each group were continuously irradiated with 808 nm laser (0.5
mW/cm.sup.2) for 5 min. Firstly, to verify that TPA-T-TQ ONP is
able to generate heat with laser irradiation in living mice, the
tumor temperatures of "ONPs+Laser"-treated and
"Saline+Laser"-treated mice were monitored by IR thermography at
different laser irradiation time scales.
[0142] As depicted in FIGS. 12A and 12B, the tumors from mice in
the "Saline+Laser" group exhibit little temperature elevation
(.DELTA.T.about.2.5.degree. C.) upon NIR light exposure for 5 min,
implying that laser irradiation alone would have negligible effect
on heat-caused tumor inhibition. In comparison, fast temperature
elevation is observed from the tumors in the "ONPs+Laser"-treated
mice, as evidenced by the tumor temperature raising from 36.degree.
C. to a plateau of about 64.degree. C. in 3 min light exposure.
Such in vivo temperature rise rate and elevated temperatures are
comparable to the best light-to-heat conversion performances
achieved by currently available organic photothermal agents. These
results illuminate that the TPA-T-TQ ONPs can result in a rapid
temperature elevation in a tumor site under NIR light irradiation,
representing an efficient agent for tumor PTT in living
organisms.
[0143] The in vivo antitumor efficacy of "ONPs+Laser" through a
single PTT was investigated by monitoring the tumor volumes for 16
days. As presented in FIG. 12C, the treatment of the "Saline+Laser"
group totally fails to suppress the tumor growth as compared to the
control group ("Only Saline"), indicating that pure 808 nm laser
irradiation does not possess any antitumor effect. Moreover, the
tumor growth kinetics from mice in the "Only ONPs" group is also
similar to that in the "Only Saline" group, suggesting that the
ONPs taken by themselves have negligible active behavior against
cancer. Dramatically, as compared to the other three groups with
fast-growing tumor volumes, the "ONPs+Laser" group shows amazing
antitumor efficacy. The average tumor volume on day 16 in the
"ONPs+Laser" group is even slightly smaller than that on day 0,
suggesting that the PTT by TPA-T-TQ ONPs is capable of resulting in
tumor growth stoppage, which is indeed efficacious on tumor
suppression. The mice in each treatment group were also weighed
every other day during a 16-day study duration. As shown in FIG.
12D, no obvious body weight loss is observed in mice of the "Only
ONPs", "Saline+Laser", and "ONPs+Laser" groups when compared with
the control group, suggesting the low toxic side effect of the
treatment of "ONPs+Laser".
Example 8
Histological Studies
[0144] Sixteen days after photothermal treatment, the
above-mentioned four groups of mice were sacrificed and tumors and
important normal organs were excised, sliced and stained. The
fluorescent PCNA staining was conducted following common
immunohistochemical steps. The fluorescent TUNEL staining was
conducted following manual instruction of the DeadEnd fluorometric
TUNEL system kit (Promega, USA). For hematoxylin and eosin
(H&E) staining, the tissues of the mice were fixed in 4%
formalin, processed into paraffin, and sectioned at 5 .mu.m
thickness. The slices were examined by a digital microscope (Leica
QWin) (FIG. 13A). To study whether TPA-T-TQ ONPs cause in vivo
toxicity, the livers and spleens of mice in each treatment group
were also excised and sectioned for H&E staining at the end
time point, as it is generally accepted that nanomaterials tend to
be enriched in reticuloendothelial system (RES) organs including
liver and spleen. No noticeable tissue damage and/or inflammatory
lesion were found in the liver and spleen organs from all the
treatment groups of mice (FIG. 13B).
Example 9
Serum Biochemistry Assay and Complete Blood Count
[0145] Healthy Balb/c mice were randomly divided into 2 groups (n=3
per group). 150 .mu.L of TPA-T-TQ ONPs (250 .mu.g/mL based on
TPA-T-TQ) was intravenously (i.v.) injected into one group of mice.
For the other group, no treatment was performed. After one week,
blood was collected for all mice and then detected using an
automated hematology analyzer. In order to further investigate the
potential toxicology of TPA-T-TQ ONPs, healthy Balb/c mice
intravenously administrated with TPA-T-TQ ONPs (250 .mu.g/mL based
on TPA-T-TQ) as well as untreated healthy mice received serum
biochemistry assay and complete blood count on day 7
post-injection. The liver function indicators including alanine
aminotransferase (ALT), aspartic acid transaminase (AST), albumin
(ALB), total bilirubin (TBIL), alkaline phosphatase (ALP), and
.gamma.-globulin transferase (GGT), all measured normal (FIG. 14A),
and revealed no obvious hepatic and kidney disorders of
"ONPs+Laser"-treated mice. The assay of complete blood panel
including white blood cells (WBC), lymphocyte (LYM), hematocrit
(HCT), hemoglobin (Hgb), red blood cells (RBC), red cell
distribution width (RDW), corpuscular hemoglobin concentration
(CHC), platelets (PLT), as well as mean platelet volume (MPV)
indicated that there are no statistical differences in these
indicators between ONPs and untreated groups (FIGS. 14A-14B).
Further, considering the negligible influences of TPA-T-TQ ONPs on
mouse body weight FIG. 12D and the health of important normal
organs FIG. 13B, it is reasonable to conclude that the TPA-T-TQ ONP
is a highly biocompatible phototheranostic nanoagent which induces
no noticeable side effect to living mice.
[0146] The present subject matter being thus described, it will be
apparent that the same may be modified or varied in many ways. Such
modifications and variations are not to be regarded as a departure
from the spirit and scope of the present subject matter, and all
such modifications and variations are intended to be included
within the scope of the following claims.
* * * * *